Induction of Apoptosis by Cellular Stress

ABSTRACT

The invention provides methods of screening to identify compounds that modulate the ability of a protein to translocate to the mitochondria when a cell is subjected to cellular stress. Such compounds can be useful to modulate the level of apoptosis in a cell. For example, compounds identified according to the methods described herein can be used to treat disorders characterized by excessive apoptosis, e.g., a neurological disorder, or insufficient apoptosis, e.g., cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/284,785, filed Apr. 18, 2001. The entire content of the priorapplication us incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant numberCA42802 awarded by the National Cancer Institute. The Government mayhave certain rights in the invention.

FIELD OF THE INVENTION

The invention relates apoptosis, and in particular to the identificationof compounds that modulate the translocation of proteins to themitochondria upon the induction of cellular stress.

BACKGROUND OF THE INVENTION

Normal cellular metabolism is associated with the production of reactiveoxygen species (ROS) and, as a consequence, damage to DNA and proteins.ROS have been implicated as signaling molecules that contribute toneurodegenerative diseases and aging. The generation of ROS isassociated with apoptosis and certain cells, particularly neurons, arehighly sensitive to this response. Studies have indicated thatROS-induced apoptosis is p53-dependent and that p53-induced apoptosis ismediated by ROS. In addition, the p66shc adaptor protein and the p85subunit of phosphatidylinositol 3-kinase (PI3-K) have been implicated inthe apoptotic response to oxidative stress.

Antioxidants have been proposed as one approach to diminish ROS damageto cells. Importantly, the signaling pathways responsible forROS-induced apoptosis are for the most part unknown.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery that, when acell is subjected to a cellular stress, proteins such as protein kinaseC delta (PKCd) and c-Abl are translocated into the mitochondria as partof the apoptotic program that eventually leads to cell death. Thepresent invention provides methods of screening to identify compoundsthat modulate the ability of a protein to translocate to themitochondria when a cell is subjected to cellular stress. Compoundsidentified according to the methods described herein can be used totreat disorders characterized by excessive apoptosis, e.g., aneurological disorder, or insufficient apoptosis, e.g., cancer.

The invention is also based, at least in part, on the discovery that theprotein catalase is ubiquitinated and that the ubiquitination status andstability of catalase is regulated by its phosphorylation by c-Abl andArg (a nonreceptor tyrosine kinase that has an overall structure similarto that of c-Abl). Catalase is a major effector in the regulation ofintracellular ROS levels (see, e.g., Linton et al. (2001) Exp. Gerontol.36:1503-18). In particular, catalase is an endogenous antioxidant enzymethat protects a cell from oxidative damage by degrading intracellularH₂O₂. Accordingly, the compositions and methods described herein can beused to modulate catalase levels and/or activity in a cell and thereforemodulate ROS levels in the cell as well as the associated ROS-inducedcell death.

In one aspect, the invention features a method of identifying a compoundthat inhibits mitochondrial translocation of a protein. The methodincludes the steps of: providing a cell; subjecting the cell to acellular stress, wherein the cellular stress induces mitochondrialtranslocation of the protein; contacting the cell with a test compound;and determining whether mitochondrial translocation of the protein isdecreased when the cell is contacted with the test compound, thedecrease being an indication that the test compound inhibitsmitochondrial translocation of the protein. The protein can be anyprotein, e.g., a protein kinase such c-Abl or PKCd, that translocates tothe mitochondria upon the induction of cellular stress.

“Mitochondrial translocation of a protein” refers to the migration of aprotein into the mitochondria from a location in the cell but outside ofthe mitochondria, e.g., from the cytoplasm, nucleus, or endoplasmicreticulum (ER). Mitochondrial translocation does not require that allspecies of a particular protein in a cell be translocated into themitochondria. For example, mitochondrial translocation can includetranslocation of all or a portion of the species of a protein located inone compartment, e.g., the ER, whereas no translocation from anothercompartment, e.g., the nucleus, is detected.

A “test compound” can be any compound, synthetic or naturally occurring.Examples of test compounds, include but are not limited to peptides,polypeptide, antibodies, and small organic or inorganic molecules.

“Cellular stress” refers to a treatment that, when applied to a cell,induces cell death. Examples of cellular stresses include oxidativestress, endoplasmic reticulum stress, cytoskeletal stress, and genotoxicstress. Cellular stress can be induced by, for example, subjecting acell to a compound, radiation, or a temperature that induces cell death.

“Oxidative stress” refers to a treatment that results in the generationof reactive oxygen species (ROS) within a cell. Examples of ROS includesinglet oxygen, hydroxyl radicals, superoxide, hydroperoxides, andperoxides. One method of subjecting a cell to oxidative stressconstitutes administering hydrogen peroxide (H₂O₂) to the cell.

“Endoplasmic reticulum stress” refers to a treatment that modulates anormal function of the ER. Generally, an ER stress results in theaccumulation of unfolded proteins in the ER. Examples of ER stressinclude treatments that increase intracellular calcium pools or blocktransport of proteins from the ER to Golgi.

“Cytoskeletal stress” refers to a treatment that modulates a normalfunction of the cytoskeleton.

“Genotoxic stress” refers to a treatment that causes damage to the DNAof a cell. Examples of agents that induce genotoxic stress includeionizing radiation and mutagenic compounds.

In one embodiment, the cellular stress includes oxidative stress. Forexample, a cell can be subjected to oxidative stress by contacting thecell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

One example of a method described herein is a method of identifying apeptide that inhibits mitochondrial translocation of c-Abl, includingthe steps of: providing a cell; contacting the cell with H₂O₂; furthercontacting the cell with a peptide; and determining whethermitochondrial translocation of c-Abl is decreased when the cell iscontacted with the peptide, the decrease being an indication that thepeptide inhibits mitochondrial translocation of c-Abl.

In another aspect, the invention features a method of identifying acompound that increases mitochondrial translocation of a protein. Themethod includes the steps of: providing a cell; contacting the cell witha test compound; and determining whether mitochondrial translocation ofthe protein is increased when the cell is contacted with the testcompound, the increase being an indication that the test compoundincreases mitochondrial translocation of the protein. The protein can beany protein, e.g., a protein kinase such c-Abl or PKCd, thattranslocates to the mitochondria upon the induction of cellular stress.

In another aspect, the invention features a method of identifying aprotein that is translocated to the mitochondria upon the induction ofcellular stress. The method includes the steps of: providing a cell;subjecting the cell to a cellular stress; and identifying a protein thatis translocated to the mitochondria of the cell. The protein can be anyprotein, e.g., a protein kinase, that translocates to the mitochondriaupon the induction of cellular stress.

In one embodiment, the cellular stress includes oxidative stress. Forexample, a cell can be subjected to oxidative stress by contacting thecell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

The step of detecting the mitochondrial translocation of the protein caninclude isolating the mitochondria of the cell and determining thepresence or amount of the protein in the mitochondria as compared to thepresence or amount of the protein in the mitochondria of a cell notsubjected to the cellular stress. In one example, the method includesdetermining the amino acid sequence of all or a part of the protein.

In another aspect, the invention features a method of identifying aprotein that interacts with a mitochondrial-translocated protein. Themethod includes the steps of: providing a cell; subjecting the cell to acellular stress, wherein the cellular stress induces the mitochondrialtranslocation of a first protein; and identifying a second protein thatbinds to the first protein in the mitochondria upon the translocation ofthe first protein to the mitochondria. The first protein can be anyprotein, e.g., a protein kinase such as PKCd or c-Abl, that translocatesto the mitochondria upon the induction of cellular stress.

In one embodiment, the cellular stress includes oxidative stress. Forexample, a cell can be subjected to oxidative stress by contacting thecell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

The identifying step can include isolating the first protein anddetecting the second protein bound to the first protein. For example,the method can include determining the amino acid sequence of all or apart of the second protein.

In another aspect, the invention features a method of identifying acompound that inhibits a protein-protein interaction. The methodincludes the steps of: providing a biological sample containing a firstprotein and a second protein, wherein the first protein is a proteinthat translocates to the mitochondria upon the induction cellularstress, and wherein the second protein interacts with the first proteinwhen the first protein translocates to the mitochondria; contacting thebiological sample with a test compound; and determining whether thefirst protein and the second protein interact in the presence of thetest compound, wherein a decreased interaction between the first andsecond proteins in the presence of the test compound indicates that thecompound inhibits the interaction. The first protein can be any protein,e.g., a protein kinase such as PKCd or c-Abl, that translocates to themitochondria upon the induction of cellular stress.

In one embodiment, the cellular stress includes oxidative stress. Forexample, a cell can be subjected to oxidative stress by contacting thecell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

In another aspect, the invention features a composition that binds toc-Abl or PKCd and inhibits cellular stress-induced mitochondrialtranslocation of c-Abl or PKCd.

The cellular stress can include oxidative stress. For example, a cellcan be subjected to oxidative stress by contacting the cell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

In another aspect, the invention features a composition that binds toc-Abl or PKCd and increases cellular stress-induced mitochondrialtranslocation of c-Abl or PKC.

The cellular stress can include oxidative stress. For example, a cellcan be subjected to oxidative stress by contacting the cell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

In another aspect, the invention features a method of inhibitingapoptosis of a cell, the method including inhibiting mitochondrialtranslocation of a protein in the cell. The protein can be any protein,e.g., a protein kinase such as PKCd or c-Abl, that translocates to themitochondria upon the induction of cellular stress.

In one embodiment, the cellular stress includes oxidative stress. Forexample, a cell can be subjected to oxidative stress by contacting thecell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

In another aspect, the invention features a method of increasingapoptosis of a cell, the method including increasing mitochondrialtranslocation of a protein in the cell. The protein can be any protein,e.g., a protein kinase such as PKCd or c-Abl, that translocates to themitochondria upon the induction of cellular stress.

In one embodiment, the cellular stress includes oxidative stress. Forexample, a cell can be subjected to oxidative stress by contacting thecell with H₂O₂.

In another embodiment, the cellular stress includes ER stress. Forexample, a cell can be subjected to ER stress by administering asubstance to the cell that increases intracellular calcium pools orblocks transport of proteins from the ER to Golgi.

In another embodiment, the cellular stress includes cytoskeletal stress.

In another embodiment, the cellular stress includes genotoxic stress.

In another aspect, the invention features a method of treatment. Themethod includes the following steps: selecting an individual sufferingfrom or at risk of contracting a disorder associated with inappropriatelevels of apoptosis; and administering to the individual a compound thatmodulates the mitochondrial translocation of c-Abl or PKCd. In oneexample, the disorder is characterized by excessive apoptosis, e.g., aneurological disorder. In another example, the disorder is characterizedby insufficient apoptosis, e.g., cancer.

In another aspect, the invention features a method for identifying acompound that modulates (increases or decreases) binding of catalase toc-Abl or Arg, the method including: a) measuring binding of a firstpolypeptide containing catalase to a second polypeptide containing c-Ablor Arg in the presence of a test compound; and b) comparing the bindingof the first polypeptide to the second polypeptide measured in step (a)to the binding of the first polypeptide to the second polypeptide in theabsence of the test compound, wherein altered binding of the firstpolypeptide to the second polypeptide in the presence of the testcompound compared the binding in the absence of the test compoundindicates that the test compound modulates the binding of catalase toc-Abl or Arg.

The first polypeptide can optionally include only a fragment ofcatalase, e.g., human catalase, that binds to c-Abl or Arg. As describedherein, c-Abl and Arg bind to the PFNP motif of catalase. Accordingly,the first polypeptide preferably contains the amino acid sequence PFNP.The first polypeptide can also include a fragment of catalase containingan amino acid residue, e.g., a tyrosine residue, that is phosphorylatedby c-Abl and/or Arg. For example the first polypeptide can containtyrosine 231 and/or tyrosine 386 of catalase.

The second polypeptide can optionally include only a fragment of c-Ablor Arg, e.g., human c-Abl or Arg, that binds to catalase. For example,the second polypeptide can include all or a portion of an SH3 domain ofc-Abl or Arg that mediates binding to catalase.

The screening method can optionally include an additional step ofmeasuring the binding of the first polypeptide to the second polypeptidein the absence of the test compound.

The screening method can be carried out in a cell-based system and/or ina cell-free system. For example, the binding of the first and secondpolypeptides can be measured in vitro using purified polypeptides in acell free system. In addition, the ability of a test compound tomodulate the binding of the first polypeptide to the second polypeptidecan be measured on a living cell, using a cell-based system. In a cellbased system, cells used in a screen can be recombinantly produced thatexpress catalase, c-Abl, Arg, and/or a fragment of any of these proteinsas described herein.

In another aspect, the invention features a method for identifying amodulator (activator or inhibitor) of catalase phosphorylation, themethod including: a) contacting a first polypeptide containing catalaseto a second polypeptide containing c-Abl or Arg in the presence of atest compound; b) measuring phosphorylation of the first polypeptide inthe presence of the test compound, wherein altered phosphorylation ofthe first polypeptide in the presence of the test compound compared tothe absence of the test compound indicates that the compound is amodulator of catalase phosphorylation.

The first polypeptide can optionally include only a fragment ofcatalase, e.g., human catalase, that is subject to phosphorylation byc-Abl or Arg. As described herein, c-Abl and Arg bind to the PFNP motifof catalase and phosphorylates tyrosine residue number 231 and tyrosineresidue number 386 of catalase. Accordingly, the first polypeptidepreferably contains the amino acid sequence PFNP. The first polypeptidecan also include a fragment of catalase containing tyrosine 231 and/ortyrosine 386 of catalase.

The second polypeptide can optionally include only a fragment of c-Ablor Arg, e.g., human c-Abl or Arg, that phosphorylates catalase. Forexample, the second polypeptide can include all or a portion of a kinasedomain of c-Abl or Arg that mediates phosphorylation of catalase. Thesecond polypeptide can also include an SH3 domain of c-Abl or Arg thatmediates binding to catalase.

The screening method can optionally include an additional step ofmeasuring the phosphorylation of the first polypeptide in the absence ofthe test compound.

The screening method can be carried out in a cell-based system and/or ina cell-free system. For example, the phosphorylation of the firstpolypeptide can be measured in vitro using purified polypeptides in acell free system. In addition, the ability of a test compound tomodulate the phosphorylation of the first polypeptide can be measured ona living cell, using a cell-based system. In a cell based system, cellsused in a screen can be recombinantly produced that express catalase,c-Abl, Arg, and/or a fragment of any of these proteins as describedherein.

In another aspect, the invention features a method for identifying amodulator (activator or inhibitor) of catalase ubiquitination, themethod including: a) contacting a polypeptide containing catalase toubiquitin in the presence of a test compound; b) measuring theubiquitination of the polypeptide in the presence of the test compound,wherein altered ubiquitination of the polypeptide in the presence of thetest compound compared to the absence of the test compound indicatesthat the compound is a modulator of catalase ubiquitination.

The polypeptide can optionally include only a fragment of catalase,e.g., human catalase, that binds to ubiquitin. The polypeptide can alsooptionally include a fragment of catalase that binds to c-Abl or Argand/or is subject to phosphorylation by c-Abl or Arg, as describedherein.

The screening method can optionally include an additional step ofmeasuring the ubiquitination of the polypeptide in the absence of thetest compound.

The screening method can be carried out in a cell-based system and/or ina cell-free system. For example, the ubiquitination of the polypeptidecan be measured in vitro using purified polypeptides in a cell freesystem. In addition, the ability of a test compound to modulate theubiquitination of the polypeptide can be measured on a living cell,using a cell-based system. In a cell based system, cells used in ascreen can be recombinantly produced that express catalase or a fragmentthereof as described herein and optionally c-Abl, Arg, and/or a fragmentof any of these proteins as described herein.

In another aspect, the invention features a method for identifying amodulator (activator or inhibitor) of catalase phosphorylation, themethod including: a) contacting a polypeptide containing catalase to atest compound; b) measuring phosphorylation of the polypeptide in thepresence of the test compound, wherein altered phosphorylation of thepolypeptide in the presence of the test compound compared to the absenceof the test compound indicates that the compound is a modulator ofcatalase phosphorylation.

The polypeptide can optionally include only a fragment of catalase,e.g., human catalase, that is subject to phosphorylation. For example,the first polypeptide can contain the amino acid sequence PFNP and/or afragment of catalase containing tyrosine 231 and/or tyrosine 386 ofcatalase.

The screening method can optionally include an additional step ofmeasuring the phosphorylation of the polypeptide in the absence of thetest compound.

The screening method can be carried out in a cell-based system and/or ina cell-free system. For example, the phosphorylation of the polypeptidecan be measured in vitro using purified polypeptides in a cell freesystem. In addition, the ability of a test compound to modulate thephosphorylation of the polypeptide can be measured on a living cell,using a cell-based system. In a cell based system, cells used in ascreen can be recombinantly produced that express catalase or a fragmentthereof as described herein and optionally c-Abl, Arg, and/or a fragmentof any of these proteins as described herein.

In another aspect, the invention features a method of modulating(increasing or decreasing) apoptosis of a cell, the method includingmodulating the phoshorylation status of catalase in the cell.

In another aspect, the invention features a method of modulating(increasing or decreasing) apoptosis of a cell, the method including theubiquitination status of catalase in the cell.

In another aspect, the invention features a method of treatment, themethod including: selecting an individual suffering from or at risk ofcontracting a disorder associated with inappropriate levels ofapoptosis; and administering to the individual an amount of a compoundsufficient to modulate the phoshorylation status of catalase.

In another aspect, the invention features a method of treatment, themethod including: selecting an individual suffering from or at risk ofcontracting a disorder associated with inappropriate levels ofapoptosis; and administering to the individual an amount of a compoundsufficient to modulate the ubiquitination status of catalase.

Unless otherwise defined; all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

The present invention provides methods of screening to identifycompounds that modulate the ability of a protein to translocate to themitochondria when a cell is subjected to cellular stress. Such compoundscan be useful to modulate the level of apoptosis in a cell. For example,compounds identified according to the methods described herein can beused to treat disorders characterized by excessive apoptosis, e.g., aneurological disorder, or insufficient apoptosis, e.g., cancer.

As described in the accompanying Examples, mitochondrial signalingpathway has been identified that is activated in the response of a cellto cellular stress. For example, the protein kinases PKCd and c-Abl areshown to be activated by ROS exposure, target the mitochondria, andcontribute to ROS-induced apoptosis. In this context, the blockade ofthese pathways results in a substantially complete abrogation ofROS-induced apoptosis (see Examples). By use of screening assaysdescribed herein, compounds can be identified that either inhibit orincrease this mitochondrial translocation event and thereby inhibit orincrease the level of apoptosis in a cell.

Methods of Screening

The present invention includes methods of screening to identifycompounds that modulate (increase or decrease) the mitochondrialtranslocation of a protein, e.g., a protein kinase such as PKCd orc-Abl. In general, such compounds can be identified by subjecting a cellto a cellular stress that induces the mitochondrial translocation of aprotein and determining whether a test compound is capable of modulatingthe mitochondrial translocation of the protein. Examples of cellularstress include oxidative stress, endoplasmic reticulum stress,cytoskeletal stress, and genotoxic stress.

Compounds that may be screened in accordance with the invention include,but are not limited to peptides, antibodies and fragments thereof, andother organic compounds that bind to a mitochondrial-translocatedprotein and modulate its activity, e.g., its ability to translocate tothe mitochondria. Examples of such compounds include: peptides such assoluble peptides, including members of random peptide libraries andcombinatorial chemistry-derived molecular libraries made of D- and/or Lconfiguration amino acids; phosphopeptides (e.g., members of random orpartially degenerate, directed phosphopeptide libraries); antibodies(e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric orsingle chain antibodies, and FAb, F(ab′)₂ and FAb expression libraryfragments, and epitope-binding fragments thereof); and small organic orinorganic molecules. Compounds that can be screened in accordance withthe invention include small organic molecules that are able to gainentry into an appropriate cell and affect the ability of a protein,e.g., PKCd or c-Abl, to translocate to the mitochondria.

Computer modeling and searching technologies can be used to assist inthe identification of compounds that can modulate the ability of aprotein to translocate to the mitochondria. The active site of acompound can be identified using methods known in the art including, forexample, the study of complexes of the relevant compound or compositionwith its ligand, e.g., PKCd or c-Abl.

In general, the screening methods described herein entail the detectionof the presence or the amount of a protein, e.g., PKCd or c-Abl, in themitochondria of a cell subjected to a particular cellular stress. Thepresence or amount of the protein in the mitochondria, as compared to anuntreated cell, is indicative of the protein's translocation to themitochondria. This mitochondrial translocation event can be detected bya variety of methods well known to those of skill in the art. Forexample, a particular protein to be analyzed, e.g., PKCd or c-Abl, canbe linked to a detectable marker, e.g., green fluorescent protein, andthe presence of the detectable marker in the mitochondria can be used anindication of the presence protein in the mitochondria. In addition,cellular components can be separated and the mitochondria can beanalyzed for the presence or amount of a particular protein. Thus,according to these methods, the ability of a protein to translocate tothe mitochondria in the presence of a test compound can be evaluated.

Compounds identified via methods described herein may be useful, forexample, for the treatment of disorders associated with inappropriatelevels of apoptosis or aberrant activity or expression of amitochondrial-translocated protein.

Identification of Molecules that Interact withMitochondrial-Translocated Proteins

The invention features methods for identifying molecules that canassociate with mitochondrial-translocated proteins. Themitochondrial-translocated protein can be any protein translocated tothe mitochondria in response to a cellular stress, e.g., a cellularstress as described herein. Examples of mitochondrial-translocatedproteins include protein kinases such as c-Abl and PKCd.

Any method that is suitable for detecting an interaction, e.g., aprotein-protein interaction, between two molecules can be employed todetect a molecule that associates with a mitochondrial-translocatedprotein. For example, the method includes identifying a protein, e.g., anaturally occurring protein, that interacts with amitochondrial-translocated protein when the mitochondrial-translocatedprotein is translocated to the mitochondria. Among the traditionalmethods that can be employed are co-immunoprecipitation, crosslinking,and co-purification through gradients or chromatographic columns of celllysates or proteins obtained from cell lysates and the use of themitochondrial-translocated protein to identify proteins in the lysatethat interact with the mitochondrial-translocated protein. For theseassays, the mitochondrial-translocated protein can be a full length or afragment of the protein, e.g., a fragment containing a catalytic domainsuch as a kinase domain. A protein associated with a mitochondrialtranslocated protein can be identified by, for example, use of twodimensional gel analysis and/or mass spectrometry (e.g., a techniquesuch as Matrix Assisted Laser Desorption/Ionization (MALDI) massanalysis). For example, proteins that bind to themitochondrial-translocated protein and are selectively detected by gelelectrophoresis after the cell is subjected to cellular stressconstitute candidates for sequencing by mass spectrometry methods. Onceisolated, such an interacting protein can be identified and cloned andthen used, in conjunction with standard techniques, to alter theactivity of the mitochondrial-translocated protein with which itinteracts. For example, the interacting protein can be used in ascreening assay of a type described herein to identify a compound thatmodulates the protein-protein interaction and thus modulates theapoptotic pathway.

Identification of Mitochondrial-Translocated Molecules

The present application describes a variety of pathways that involve thetranslocation of proteins to the mitochondria following the treatment ofa cell with a cellular stress. This identification of the mitochondriaas a site of protein translocation as a part of the apoptotic programpermits the use of the organelle to screen for additional molecules(including proteins as well as any other molecules) that translocate,either in or out, of the mitochondria in response to cellular stress.Such molecules constitute important targets for the generation ofcompounds that modulate apoptosis.

Mitochondrial-translocated molecules can be identified by using methodssimilar to those of other screening assays described herein. Forexample, the mitochondrial protein content of a cell subjected to acellular stress can be compared to the mitochondrial protein content acell not subjected to the stress. A protein that is detected aspreferentially translocating into or out of the mitochondria in responseto a cellular stress constitutes a candidate for furthercharacterization. For example, a mitochondrial-translocated protein canbe initially identified by two dimensional gel electrophoresis (e.g., asa protein present in the mitochondria of stressed cells but not incontrol cells) and then further characterized by mass spectrometry(e.g., by MALDI mass analysis).

Profiling of proteins and/or other molecules (such as co-factors ormetabolites) in the mitochondria pre and post stress induction, e.g.,oxidative stress induction, can provide comprehensive understanding oftranslocations in and out of the mitochondria under stress conditions.Such profiling screens can be performed in any cell type, e.g., tumorcell lines or neuronal cell lines. Stress inducing agents include thosethat induce oxidative stress, endoplasmic reticulum stress, cytoskeletalstress, and genotoxic stress.

Methods of Treatment

As described herein, modulating (increasing or decreasing) themitochondrial translocation of a protein such as c-Abl or PKCd can beused to treat an individual suffering from or at risk of contracting adisorder associated with inappropriate levels of apoptosis. Compositionsuseful for modulating mitochondrial translocation of c-Abl or PKCd aredescribed herein.

Certain disorders are associated with an increased number of survivingcells that are produced and continue to survive or proliferate whenapoptosis is inhibited. These disorders include, for example, cancer(particularly follicular lymphomas, carcinomas associated with mutationsin p53, and hormone-dependent tumors such as breast cancer, prostatecancer, and ovarian cancer), autoimmune disorders (such as systemiclupus erythematosis, immune-mediated glomerulonephritis), and viralinfections (such as those caused by herpesviruses, poxviruses, andadenoviruses). For example, failure to remove autoimmune cells thatarise during development or that develop as a result of somatic mutationduring an immune response can result in autoimmune disease.

Populations of cells are often depleted in the event of viral infection,with perhaps the most dramatic example being the cell depletion causedby the human immunodeficiency virus (HIV). Surprisingly, most T cellsthat die during HIV infections do not appear to be infected with HIV.Although a number of explanations have been proposed, recent evidencesuggests that stimulation of the CD4 receptor results in the enhancedsusceptibility of uninfected T cells to undergo apoptosis.

A wide variety of neurological diseases are characterized by the gradualloss of specific sets of neurons. Such disorders include Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),retinitis pigmentosa, spinal muscular atrophy, and various forms ofcerebellar degeneration. The cell loss in these diseases does not inducean inflammatory response, and apoptosis appears to be the mechanism ofcell death.

In addition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses.

Two common disorders associated with cell death are myocardialinfarctions and stroke. In both disorders, cells within the central areaof ischemia, which is produced in the event of acute loss of blood flow,appear to die rapidly as a result of necrosis. However, outside thecentral ischemic zone, cells die over a more protracted time period andmorphologically appear to die by apoptosis.

Pharmaceutical Compositions

Compounds that modulate the mitochondrial translocation of a proteinsuch as c-Abl or PKCd are expected to be useful in modulating the celldeath that is associated with this translocation event. Methods ofidentifying a variety of compounds that modulate mitochondrialtranslocation of a protein are described herein. These compounds can beused to treat disorders characterized by excessive or insufficientapoptosis.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in a conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by inhalation, insufflation (eitherthrough the mouth or the nose), oral, buccal, parenteral or rectaladministration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc, or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate).Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, for example, containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (e.g., subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

The therapeutic compositions of the invention can also contain a carrieror excipient, many of which are known to persons of ordinary skill inthe art. Excipients that can be used include buffers (e.g., citratebuffer, phosphate buffer, acetate buffer, and bicarbonate buffer), aminoacids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g.,serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol,and glycerol.

EXAMPLES Example 1 Activation of the Cytoplasmic c-Abl Tyrosine Kinaseby Reactive Oxygen Species

Cell culture: COS7 cells and mouse embryonic fibroblasts (MEFs) derivedfrom wild-type and c-Abl^(−/−) mice were cultured in Dulbecco's modifiedEagle's medium containing 10% heat-inactivated fetal calf serum, 2 mML-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. DLD1cells were grown as described (Polyak et al. (1997) Nature 389,300-305). Cells were treated with H₂O₂ (Sigma), 30 mMN-acetyl-L-cysteine (NAC; Sigma) or 10 μM cis-platinum (Sigma).

Analysis of kinase activity: Cell lysates were prepared in lysis buffer(10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 3 mM MgCl₂,0.5 mM PMSF, 5 μg/ml leupeptin) containing 0.5% Nonidet P-40 andsubjected to immunoprecipitation as described (Kharbanda et al. (1997)Nature 386, 732-735) with anti-c-Abl (sc-23; Santa Cruz) or mouse IgG(Santa Cruz). The immunoprecipitates were resuspended in kinase buffer(20 mM HEPES, pH 7.4, 10 mM MgCl₂, 10 mM MnCl₂) containing 2.5 μCi[g-32P]ATP and GST-Crk(120-225) or GST-Crk(120-212) for 20 minutes at30° C. Anti-PKCd (sc-937; Santa Cruz) and anti-ERK1 (sc-93; Santa Cruz)immunoprecipitates were analyzed by using histone H1 and myelin basicprotein (Upstate Biotecnology Inc.), respectively, as substrates. Thereaction products were analyzed by SDS-PAGE and autoradiography.

Isolation of cytoplasmic and nuclear fractions: Cells were disrupted inlysis buffer containing 0.05% NP-40. The cytoplasmic and nuclearfractions were prepared as described (Kharbanda et al. (1996) CancerRes. 56, 3617-3621).

Preparation of cytoplasts: Enucleated cells were prepared by densitycentrifugation as described (Gudas et al. (1986) J. Cell. Physiol. 128,441-448). Cells were incubated in 21 μM cytochalasin B for 1 hour at 37°C., layered over a discontinuous Ficoll gradient and centrifuged at80,000×g for 1 hour. Cytoplasts were collected at the 12.5-15% Ficollinterface. Cytoplast purity was assessed by staining with 0.5 mg/ml4′,6-diamino-2-phenylindole (DAPI) and was greater than 95% free ofwhole cells.

Immunoblot analysis: Proteins were separated by SDS-PAGE, transferred tonitrocellulose and probed with anti-c-Abl, anti-IkBα (sc-847; SantaCruz) or anti-cytochrome c (Kirken et al. (1995) Protein Expression &Purification 6, 707-715). The antigen-antibody complexes were visualizedby enhanced chemiluminescence (ECL; Amersham).

Apoptosis assays: DNA content was assessed by staining ethanol-fixedcells with propidium iodide and monitoring by FACScan(Becton-Dickinson).

Results and Discussion:

To determine whether c-Abl is activated by ROS, lysates from COS7 cellsexposed to H₂O₂ were subjected to immunoprecipitation with mouse IgG, asa control, or anti-c-Abl antibody. The precipitates were assayed forphosphorylation of a GST-Crk(120-225) fusion protein (Feller et al.(1994) EMBO J. 13, 2341-2351; Ren et al. (1994) Genes Dev. 8, 783-795).There was no detectable phosphorylation of GST-Crk(120-225) with thecontrol immunoprecipitates. A low level of GST-Crk(120-225)phosphorylation was detectable when assaying anti-c-Ablimmunoprecipitates from control cells, whereas exposure to H₂O₂ resultedin stimulation (4-5 fold) of the Crk kinase activity. By contrast, therewas no detectable H₂O₂-induced phosphorylation of a GST-Crk(120-212)fusion protein that lacks the c-Abl phosphorylation site at Y-221. Theresults also show that H₂O₂ treatment is not associated with increasesin the level of c-Abl protein. To confirm involvement of ROS in c-Ablactivation, cells were treated with N-acetylcysteine (NAC), a scavengerof reactive oxygen intermediates and precursor of glutathione (Roedereret al. (1990) Proc. Natl. Acad. Sci. (U.S.A). 87, 4884-4888; Staal etal. (1990) Proc. Natl. Acad. Sci. (U.S.A). 87, 9943-9947). NAC treatmentinhibited H₂O₂-induced phosphorylation of GST-Crk(120-225) by c-Abl. Theinduction of c-Abl activity was dependent on H₂O₂ concentration, withincreases of 5-fold upon exposure to 1 mM. In addition, maximalinduction of c-Abl activity was observed at 30-60 minutes. The findingthat human DLD1 cells respond to H₂O₂ with activation of c-Abl furtherindicated that the results are not restricted to certain cell types.

To extend the analysis of H₂O₂-induced activation of c-Abl to otherpathways involved in the ROS response, mouse embryo fibroblasts null forc-Abl expression were studied (c-Abl−/− MEFs) (Tybulewicz et al. (1991)Cell 65, 1153-1163). There was no detectable c-Abl activity is controlor H₂O₂-treated c-Abl^(−/−) cells. By contrast, wild-type MEFs respondedto H₂O₂ with induction of c-Abl activity. Recent studies havedemonstrated that c-Abl interacts with PKCd in the response to oxidativestress (Sun et al. (2000) J. Biol. Chem. 275, 7470-7473). To determinewhether c-Abl is required for activation of PKCd, we assayed anti-PKCdimmunoprecipitates from c-Abl^(−/−) and wild-type MEFs. The resultsdemonstrate that, while PKCd is required for activation of c-Abl, c-Ablis dispensable for activation of PKCd in the ROS response. Other studieshave demonstrated that ERK1 is activated in cells exposed to H₂O₂(Guyton et al. (1996) J. Biol. Chem. 271, 4138-4142). Analysis ofanti-ERK1 immunoprecipitates from H₂O₂-treated c-Abl^(−/−) and wild-typeMEFs demonstrated activation of ERK1 by a c-Abl-independent mechanism.These findings demonstrate that activation of c-Abl in the ROS responseis not functional in the induction of PKCd or ERK1 activities.

As nuclear c-Abl is activated in the stress response to DNA damage(Kharbanda et al. (1995) Nature 376, 785-788), studies were performed todefine the subcellular localization of ROS-induced c-Abl activation.Cells were treated with H₂O₂ before preparation of nuclear andcytoplasmic fractions. Analysis of cytoplasmic anti-c-Ablimmunoprecipitates demonstrated increased phosphorylation ofGST-Crk(120-225). By contrast, there was no detectable activation ofc-Abl in the nuclear fraction. Oxygen radicals induce lesions in nuclearDNA (Imlay et al. (1988) Science 240, 1302-1309; Imlay et al. (1988)Science 240, 640-642) and nuclear c-Abl is activated by DNA damage(Kharbanda et al. (1995) Nature 376, 785-788). To determine whether anuclear signal is required for H₂O₂-induced activation of cytoplasmicc-Abl, cytoplasts devoid of nuclei were assayed. Treatment of thecytoplasts with H₂O₂ was associated with induction of c-Abl activity. Bycontrast, cisplatin treatment, which activates nuclear c-Abl, had nodetectable effect on c-Abl activity in cytoplasts. These findingsindicate that cytoplasmic c-Abl is activated in the response tooxidative stress by a mechanism independent of nuclear signals.

The cellular response to genotoxic stress includes release ofmitochondrial cytochrome c and the induction of apoptosis (Kharbanda etal. (1997) Proc. Natl. Acad. Sci. USA 94, 6939-6942). To determinewhether oxidative stress induces cytochrome c release, cytoplasmiclysates from wild-type and c-Abl^(−/−) cells treated with H₂O₂ weresubjected to immunoblotting with anti-cytochrome c. The resultsdemonstrate that H₂O₂ treatment of wild-type MEFs is associated withincreased levels of cytochrome c. By contrast, cytochrome c release wasnot detectable in c-Abl^(−/−) MEFs treated with H₂O₂. To determinewhether c-Abl contributes to the induction of apoptosis by oxidativestress, H₂O₂-treated MEFs were assayed for the appearance of sub-G1 DNA.The results demonstrate that, compared to wild-type MEFs, thec-Abl^(−/−) MEFs exhibit an attenuated apoptotic response to H₂O₂exposure. Analysis at 3 to 24 h of H₂O₂ exposure confirmed that cellsdeficient in c-Abl expression exhibit a defective apoptotic response.The finding that H₂O₂-induced release of cytochrome c is completelyabrogated in c-Abl^(−/−) cells indicates that the attenuated inductionof apoptosis in response to H₂O₂ is mediated by signals by a cytochromec-independent pathway. These results collectively demonstrate thatcytoplasmic H₂O₂ induces cytochrome c release and apoptosis by ac-Abl-dependent mechanism.

Oxidative cellular damage contributes to ageing (Migliaccio et al.(1999) Nature 402, 309-313) and, in the presence of acute ROS exposure,the induction of apoptosis (Buttke et al. (1994) Immunol Today 15,7-10). Previous work has shown that the nuclear c-Abl kinase isactivated in the apoptotic response of cells to genotoxic stress (see,e.g., Yuan et al. (1999) Nature 399, 814-817). Conversely, the presentstudies demonstrate that cytoplasmic, and not nuclear, c-Abl isactivated in the apoptotic response to oxidative stress. Whereas DNAdamage-induced apoptosis is mediated by activation of c-Abl and therelease of mitochondrial cytochrome c (Kharbanda et al. (1997) Proc.Natl. Acad. Sci. USA 94, 6939-6942), less is known about involvement ofmitochondrial signals in H₂O₂-induced cell death. The present resultsdemonstrate that cytochrome c release is also induced in response tooxidative stress and that this event is c-Abl dependent. These findingssupport a model in which c-Abl functions in determining cell fate byconferring stress-induced signals to the release of cytochrome c andthereby apoptosis. The findings further indicate that the subcellulardistribution of c-Abl determines localization of the specific responseto apparently diverse environmental stresses. The present findingsdemonstrate that, analogous to activation of nuclear c-Abl by DNAdamaging agents, cytoplasmic c-Abl is activated by ROS-induced stress.

Example 2 Mitochondrial Translocation of Protein Kinase C Delta inPhorbol Ester-Induced Cytochrome C Release and Apoptosis

Cell culture and reagents: Human U-937 myeloid leukemia cells (ATCC,Rockville, Md.) were grown in RPMI 1640 medium supplemented with 10%heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100μg/ml streptomycin, and 2 mM L-glutamine. MCF-7, MCF-7/neo, MCF-7/PKCdRDand 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% FBS. Cells (3×10⁶/150 mm culture dish) were plated 24hours before treating with 250 nM TPA (Sigma Chemical Co.), 100 nMbryostatin 1 (ICN, Ohio), 10 μM 1,2-dioctanoyl-sn-glycerol (DOG;Calbiochem) and 0.5 units/ml phospholipase C (PLC; Sigma). Cells werealso treated with 10 μM rottlerin (Calbiochem).

Isolation of mitochondria: Cells were washed twice with phosphate buffersaline (PBS), homogenized in buffer A (210 mM manitol, 70 mM sucrose, 5mM HEPES, 1 mM EGTA) and 110 ug/ul digitonin in a glass homogenizer(Pyrex no. 7727-07) and centrifuged at 5000 g for 20 min. Pellets wereresuspended in buffer A, homogenized in a small glass homogenizer (Pyrexno. 7726) and centrifuged at 2000 g for 5 minutes. Supernatant (S1) wascollected and the pellet again homogenized in of buffer A. Supernatant(S2) was collected after centrifugation at 2000 g for 5 minutes.Supernatants S1 and S2 were mixed and centrifuged at 11000 g for 10 min.Mitochondrial pellets were disrupted in lysis buffer at 4° C. for 30minutes and then centrifuged at 15000 g for 20 minutes. Theconcentration of mitochondrial proteins in the supernatant wasdetermined using Bio-Rad protein estimation kit.

Isolation of the cytosolic fraction: Cells were washed twice with PBSand the pellet was suspended in 5 ml of ice-cold buffer B containing 250mM sucrose. The cells were homogenized by disrupting three times in aDounce homogenizer in buffer B (20 mM HEPES, pH 7.5, 1.5 mM MgCl₂, 10 mMKCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF and 10 μg/ml leupeptinand aprotinin). After centrifugation for 5 min at 4° C., thesupernatants then were centrifuged at 105,000×g for 30 minutes at 4° C.The resulting supernatant was used as the soluble cytosolic fraction.

Immunoprecipitation and immunoblot analysis: Total, cytoplasmic ormitochondrial lysates were subjected to immunoprecipitation withanti-GFP, anti-PKCγ (Santa Cruz Biotechnology, CA), anti-PKCμ (SantaCruz), anti-PKCζ (UBI), anti-PKCθ (Santa Cruz), anti-PKCη (Santa Cruz),anti-PKCε (Santa Cruz) or anti-PKCd (Santa Cruz) antibodies. Proteinswere separated by SDS-PAGE and transferred to nitrocellulose membranes.The residual binding sites were blocked by incubating the filters with5% nonfat dry milk in PBST (PBS/0.05% Tween 20). The filters wereincubated with anti-PKCd, anti-cytochrome c (22), anti-Hsp-60(Stressgen, Canada), anti-Actin (Sigma), anti-PKCγ, anti-PKCμ, anti-PKCζor anti-GFP (Clontech, Palo Alto, Calif.). After washing twice withPBST, the filters were incubated with anti-rabbit or anti-mouse IgGperoxidase conjugate and developed by ECL (Amersham).

Plasmids: pEGFP-PKCd and PKCd-RD were prepared as described (Kumar etal. (2000) EMBO J. 19, 1087-1097). The pEGFP-PKCd(K378R) was generatedby site-directed mutagenesis (Sun et al. (2000) J. Biol. Chem. 275,7470-7473).

Transient transfections: 293T cells were transiently transfected withempty vector (pEGFP-C1), GFP-PKCd or pEGFPCy-PKCd (K378R) usingSuperFect (Qiagen). At 24 hours after transfection, cells were lysed inlysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mMsodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mMdithiothreitol, and 10 μg/ml leupeptin and aprotinin) and subjected toimmunoblotting with anti-PKCd and anti-GFP. Signal intensities weredetermined by densitometric analysis.

Immunofluorescence microscopy: Cells immobilized on slides were fixedwith 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, incubatedwith 20 ng anti-PKCd/slide and then Texas Red-conjugated goatanti-rabbit IgG (Southern Biotechnology Associates, Inc). Mitochondriawere stained with 0.006 ng/slide of Mitotracker Green FM (MolecularProbes). The slides were analyzed using a Zeiss Auxiphot fluorescencemicroscope coupled to a CCD camera and a power Macintosh 8100. Imageanalysis was performed using the IPLab Spectrum 3.1 software (SignalAnalytics).

PKCd activity assays: 293T cells were transiently transfected withGFP-PKCd or GFP-PKCd(K—R). Total cell lysates were subjected toimmunoprecipitation with anti-PKCd, anti-PKCθ, anti-PKCε, anti-PKCη,anti-PKCμ or anti-PKCζ. The immune complex kinase assays were performedusing H1 histone as a substrate as described (Bharti et al. (1998) Mol.Cell. Biol. 18, 6719-6728).

Quantitation of apoptosis by flow cytometric analysis: Cells wereharvested, washed twice with PBS and fixed with 80% ethanol. Cells (10⁶cells/ml) were washed and incubated with propidium iodide (2.5 mg/ml)and RNase (50 mg/ml). FACScan (Becton Dickinson) was used to assesscells with sub-G1 DNA content.

Results and Discussion:

To determine whether PKC regulates mitochondrial function, human U-937cells were treated with TPA to activate PKC. PKC translocation wasassessed by subjecting cytoplasmic and mitochondrial fractions toimmunoblotting with anti-PKC antibodies. The results demonstrate thatTPA treatment is associated with decreases in cytoplasmic PKCd andconcomitant increases in mitochondrial PKCd. As controls, thecytoplasmic and mitochondrial fractions were also subjected toimmunoblotting with anti-actin and anti-Hsp60 to ensure purity of thepreparations. By contrast to translocation of PKCd, TPA had nodetectable effect on cytoplasmic or mitochondrial levels of PKCγ andPKCζ. The immunoblots were scanned to calculate percent PKCdtranslocation to mitochondria. The results demonstrate thatapproximately 40% of PKCd translocates to mitochondria in response toTPA.

The demonstration that PKCd also translocates to mitochondria inTPA-treated MCF-7 cells indicates that the finding is not restricted tocertain cell types. In addition, to confirm the subcellularredistribution of PKCd in TPA-treated cells, intracellular fluorescencewas visualized with a CCD camera and image analyzer. Examination offluorescence markers in control cells showed distinct patterns foranti-PKCd (red signal) and a mitochondrion-selective dye (Mitotracker;green signal). The demonstration that TPA induces a marked change influorescence signals (red and green→yellow/orange) supportedtranslocation of PKCd to mitochondria. These findings obtained byimmunofluorescence microscopy thus confirm the results of PKCdredistribution found by subcellular fractionation.

To determine whether the natural product bryostatin, which activates PKC(Stone et al. (1988) Blood 72, 208-213), also induces the translocationof PKCd, mitochondrial lysates from U-937 cells treated with 100 nMbryostatin were subjected to immunoblot analysis with anti-PKCd. As acontrol, mitochondrial lysates were also subjected to immunoblotanalysis with anti-PKCζ. The results demonstrate that, in contrast toPKCζ, treatment with bryostatin was associated with translocation ofPKCd to mitochondria. Phospholipase C (PLC) is activated by cellmembrane-initiated signaling pathways and, by conferring the hydrolysisof phosphatidylinositol or phosphatidylcholine, results in the formationof DAG. To determine whether PLC induces the translocation of PKCd,mitochondrial lysates from U-937 cells treated with 0.5 units/ml PLCwere subjected to immunoblot analysis with anti-PKCd. The resultsdemonstrate that, treatment with PLC is associated with translocation ofPKCd to mitochondria. To confirm the involvement of DAG in mitochondrialtranslocation of PKCd, cells were treated with a cell permeable DAG(DOG). Immunoblot analysis of DOG-treated cell lysates demonstrated thatDOG induced the translocation of PKCd to mitochondria. These findingsindicate that, like TPA, treatment with byostatin, PLC and DOG isassociated with redistribution of cytosolic PKCd to mitochondria.

To determine whether activation of PKCd is required for translocation tomitochondria, cells were transfected with a vector expressing greenfluorescence protein (GFP)-tagged PKCd. Immunoblot analysis withanti-GFP demonstrated no detectable PKCd in the mitochondrial fractionfrom cells transfected with an empty GFP vector. By contrast,transfection of kinase-active GFP-PKCd was associated with PKCdexpression in mitochondria. Moreover, treatment of theGFP-PKCd-transfected cells with TPA resulted in further increases inlevels of mitochondrial PKCd. Significantly, transfection ofkinase-inactive GFP-PKCd(K—R) had no effect on expression ofmitochondrial PKCd. In addition, overexpression of GFP-PKCd(K—R) blockedthe TPA-induced translocation of PKCd to mitochondria. To demonstratethat PKCd(K—R) specifically blocks endogenous PKCd activity, and notthat other isoforms of PKC, 293T cells were transiently transfected withGFP-PKCd or GFP-PKCd(K—R). Following transfection, cell lysates weresubjected to immunoprecipitation with anti-PKCd, anti-PKCμ, anti-PKCζ,anti-PKCθ, anti-PKCη or anti-PKCε. The precipitates were assayed in invitro kinase assays using H1 histone as substrate. The resultsdemonstrate that, in contrast to PKCμ, PKCζ, PKCθ or PKCη,overexpression of PKCd(K—R) specifically inhibits the activity ofendogenous PKCd. The results also indicate that overexpression ofPKCd(K—R) is associated with slight inhibition of the phosphorylated andactive PKCε. PKCd consists of an N-terminal regulatory domain (RD) and aC-terminal catalytically active fragment (CF) (Ghayur et al. (1996) J.Exp. Med. 184, 2399-2404). MCF-7 cells stably transfected to express the35 kDa RD exhibit attenuation of TPA-induced PKCd activity.Translocation of PKCd to mitochondria was also attenuated in TPA-treatedMCF-7/PKCdRD cells as compared to that in MCF-7 cells expressing theempty neo vector. Other studies were performed with rottlerin, aselective inhibitor of PKCd activation. Treatment of U-937 cells withrottlerin abrogated TPA-induced localization of PKCd to mitochondria.These findings collectively demonstrate that PKCd activation isnecessary for its translocation to mitochondria.

The potential role of PKCd translocation was explored by assessingmitochondrial release of cytochrome c. Whereas diverse apoptotic signalsinduce cytochrome c release, phorbol ester treatment of cells has notbeen associated with this event. Immunoblot analysis of cytoplasmicfractions with anti-cytochrome c demonstrated that TPA treatment ofU-937 cells is associated with cytochrome c release. Similar resultswere obtained when U-937 cells were treated with PLC or DOG. Todetermine whether PKCd functions in inducing cytochrome c release, U-937cells were pretreated with rottlerin before adding TPA. Of note,treatment of cells with rottlerin alone is associated with cytotoxiceffects that contribute to a detectable release of cytochrome c. Bycontrast, analysis of cytoplasmic lysates demonstrated that rottlerinsignificantly blocks TPA-induced cytochrome c release. As these findingsindicate that the PKCd kinase function is required for TPA-inducedrelease of cytochrome c, 293T cells were transfected to express GFP,GFP-PKCd or GFP-PKCd(K—R) and then treated with TPA. Immunoblotting ofthe cytoplasmic fraction from GFP positive cells demonstrated abrogationof TPA-induced cytochrome c release in cells expressing PKCd(K—R)compared to that in cells transfected with the GFP-PKCd vector. Takentogether, these results and those obtained for PKCd translocationsupport a role for PKCd in the mitochondrial release of cytochrome c.

The release of cytochrome c from mitochondria triggers activation ofcaspases and induction of apoptosis (Liu et al. (1996) Cell 86,147-157). To determine whether TPA-induced PKCd translocation andthereby cytochrome c release contributes to apoptosis, U-937 cellstreated with rottlerin and TPA were assayed for sub-G1 DNA content. Theresults demonstrate that treatment with rottlerin alone induces a lowlevel of apoptosis. By contrast, the apoptotic response of U-937 cellsto TPA was significantly attenuated by inhibition of PKCd withrottlerin. Moreover, treatment of MCF-7/neo cells with TPA was alsoassociated with the induction of apoptosis. By contrast, the apoptoticresponse to TPA was significantly attenuated in MCF-7/PKCdRD cells.Taken together with the other findings, these results support a role forTPA-induced localization of PKCd to mitochondria and in the induction ofapoptosis.

Previous work has demonstrated that TPA treatment is associated withtranslocation of PKCd to the cell membrane (Ohmori et al. (1998) Mol.cell. Biol. 18, 5263-5271). The present studies demonstrate that TPAtreatment of diverse cell types is associated with translocation of PKCdto mitochondria. These findings have been confirmed by cellfractionation and immunofluorescence studies. The results furtherdemonstrate that the PKCd kinase function is necessary for TPA-inducedmitochondrial localization. The functional significance of PKCdtranslocation to mitochondria is supported by the finding that thisevent is linked to mitochondrial release of cytochrome c. Moreover, theresults demonstrate that abrogation of PKCd translocation tomitochondria significantly inhibits TPA-induced apoptosis. Thesefindings thus support a model in which TPA induces the release ofcytochrome c and thereby apoptosis by a PKCd-dependent mechanism.

Example 3 Targeting of Protein Kinase C d to Mitochondria in theOxidative Stress Response

Cell culture and reagents: Human U-937 myeloid leukemia cells (ATCC,Manassas, Va.) were maintained in RPMI 1640 medium containing 10%heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100mg/ml streptomycin and 2 mM L-glutamine. Human MCF-7, MCF-7/neo andMCF-7/PKCdRD (15) breast cancer cells, 293T cells and wild-type andc-Abl^(−/−) MEFs (Tybulewicz et al. (1991) Cell 65, 1153-1163) weregrown in Dulbecco's modified Eagle's medium containing 10% FBS andantibiotics. Cells were treated with 1 mM H₂O₂ (Sigma Chemical Co.), 10mM rottlerin (Sigma) and 30 mM N-acetyl-L-cysteine (NAC; Calbiochem).Transfections were performed with Superfect (Qiagen).

Isolation of the cytosolic fraction: Cells were suspended in ice-cold 20mM HEPES, pH 7.5, 1.5 mM Mg Cl₂, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mMdithiothreitol (DTT), 0.1 mM phenylmethylsulphonyl fluoride (PMSF), 10mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepstatin A and 250 mMsucrose. The cells were disrupted by Douce homogenization. Aftercentrifugation at 1500×g for 5 minutes at 4° C., the supernatants werethen centrifuged at 105,000×g for 30 minutes at 4° C. The resultingsupernatant was used as the soluble cytoplasmic fraction.

Isolation of the mitochondrial fraction: Cells were suspended inice-cold 5 mM HEPES, pH 7.5, 210 mM mannitol, 1 mM EGTA, 70 mM sucroseand 110 mg/ml digitonin. The cells were disrupted in a glass homogenizer(Pyrex No. 7727-07) and centrifuged at 2000×g for 20 minutes at 4° C.The pellets were resuspended in the same buffer, homogenized again(Pyrex No. 7726) and centrifuged at 2000×g for 5 minutes at 4° C. Thesupernatants (S1) were collected. The pellets were re-homogenized,centrifuged at 2000×g for 5 minutes and the resultant supernatants (S2)collected. Supernatants S1 and S2 were pooled and centrifuged at11,000×g for 10 minutes. The mitochondrial pellets were resuspended inlysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mMsodium vanadate, 1 mM PMSF, 1 mM DTT, 10 mg/ml leupeptin and 10 mg/mlaprotinin) for 30 minutes on ice and then centrifuged at 15,000×g for 20minutes. The supernatant was used as the soluble mitochondrial fraction.Protein concentration was determined by the BioRad protein estimationkit.

Immunoblot analysis: Soluble proteins were subjected to immunoblotanalysis with anti-PKCd (Santa Cruz Biotechnology), anti-b-actin(Sigma), anti-Hsp60 (Stressgen), anti-IkBα (Santa Cruz), anti-PKCζ(Santa Cruz), anti-PKCγ (Santa Cruz), anti-GFP (Clontech) andanti-cytochrome c (Kirken et al. Prot. Exp. Purif. 6: 707-715, 1995).The immune complexes were detected with anti-rabbit or anti-mouse IgGperoxidase conjugate (Amersham) and visualized by ECL chemiluminescence(Amersham Pharmacia).

Immunofluorescence microscopy: Cells immobilized on slides were fixedwith 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, incubatedwith 20 ng of anti-PKCd/slide and then Texas Red-conjugated goatanti-rabbit IgG (Southern Biotechnology Associates). Mitochondria werestained with 0.006 ng/slide of Mitotracker Green FM (Molecular Probes).The slides were analyzed with a Zeiss Auxiphot fluorescence microscopecoupled to a CCD camera and a power Macintosh 8100. Image analysis wasperformed using the IPLab Spectrum 3.1 software (Signal Analytics).

Assessment of PKCd activity: PKCd activity was assayed by incubatinganti-PKCd immunoprecipitates in PKC kinase buffer containing 20 mMTris-HCl, pH 7.5, 10 mM Mg Cl₂, 20 mM ATP, 2.5 mCi [g-32P]ATP and 200mg/ml histone H1 for 5 minutes at 30° C. The reaction products wereanalyzed by SDS-PAGE and autoradiography.

Assessment of apoptosis: Cells were fixed with 80% ethanol, washed, andincubated with 2.5 mg/ml propidium iodide and 50 mg/ml RNase. Cells withsub-G1 DNA were determined by FACScan (Becton Dickinson).

Results and Discussion:

To assess the effects of ROS on subcellular distribution of PKCd, humanU-937 cells were treated with H₂O₂ and harvested at varying intervals.Cytoplasmic and mitochondrial fractions were subjected to immunoblottingwith anti-PKCd. The results demonstrate that treatment with 1 mM H₂O₂ isassociated with decreases in cytoplasmic and concomitant increases inmitochondrial PKCd. The finding that treatment with higherconcentrations of H₂O₂ is associated with increased localization of PKCdto mitochondria indicated that this response is dose-dependent.Immunoblot analysis of the fractions with antibodies against cytosolicb-actin and mitochondrial Hsp60 was performed to assure purity of thepreparations. In contrast to the response of PKCd to H₂O₂ treatment,there was little if any effect of this agent on mitochondrial levels ofPKCζor PKCγ. To confirm involvement of ROS in targeting of PKCd tomitochondria, cells were treated with NAC, a scavenger of reactiveoxygen intermediates and precursor of glutathione. The resultsdemonstrate that NAC inhibits H₂O₂-induced localization of PKCd tomitochondria. To extend these findings, intracellular localization ofPKCd was visualized with a CCD camera and image analyzer. Fluorescencedetection in control cells showed distinct patterns for PKCd (redsignal) and a mitochondrial-selective dye (Mitotracker; green signal).The finding that H₂O₂ treatment is associated with a change influorescence signals (red and green→yellow/orange) provided furthersupport for translocation of PKCd to mitochondria.

To determine whether PKCd activity contributes to mitochondrialtargeting of PKCd in response to H₂O₂, NIH3T3 cells were treated withthe selective PKCd inhibitor, rottlerin. As found in U-937 cells,treatment with H₂O₂ was associated with localization of PKCd tomitochondria. Moreover, the demonstration that rotterlin inhibitsH₂O₂-induced translocation of PKCd to mitochondria supported involvementof the PKCd kinase function. Other studies have demonstrated that PKCdinteracts with the c-Abl tyrosine kinase in the cellular response tooxidative stress (Sun et al. (2000) J. Biol. Chem. 275, 7470-7473). Todetermine whether c-Abl is necessary for H₂O₂-induced targeting of PKCdto mitochondria, wild-type and c-Abl^(−/−) MEFs were treated with H₂O₂.H₂O₂-induced activation of PKCd was similar in both cells. In addition,localization of PKCd to mitochondria was detectable in the response ofboth wild-type and c-Abl^(−/−) cells. To extend this analysis, cellswere transfected with vectors expressing GFP, GFP-PKCd or akinase-inactive GFP-PKCd(K378R) mutant (Sun et al. (2000) J. Biol. Chem.275, 7470-7473). Analysis of the mitochondrial fraction byimmunoblotting with anti-GFP demonstrated H₂O₂-induced targeting of PKCdto mitochondria. By contrast, H₂O₂ had no apparent effect onmitochondrial localization of GFP-PKCd(K378R). These findings indicatethat activation of the PKCd kinase function is necessary forH₂O₂-induced mitochondrial localization.

Example 1 demonstrates that treatment of COS cells with H₂O₂ isassociated with cytochrome c release. The finding that H₂O₂ also inducescytochrome c release in U-937 cells indicated that this response is notrestricted by cell type. To determine whether PKCd is functional ininducing the release of cytochrome c, U-937 cells were pretreated withNAC or rottlerin. The results demonstrate that NAC blocks H₂O₂-inducedrelease of cytochrome c. Importantly, pretreatment with rotterlin alsoblocked the release of cytochrome c in response to H₂O₂ treatment. Toextend these findings, 293 cells were transfected with GFP, GFP-PKCd orGFP-PKCd(K—R) and then treated with H₂O₂. Analysis of cytoplasmiclysates demonstrated that transfection of GFP-PKCd is associated withrelease of cytochrome c and that this effect is stimulated by H₂O₂treatment. By contrast, expression of GFP-PKCd(K378R) had no detectableeffect on cytochrome c release and blocked the response to H₂O₂. Theseresults and those obtained for translocation of PKCd to mitochondriasupport the involvement of PKCd in H₂O₂-induced release of cytochrome c.

PKCd consists of an N-terminal regulatory domain (RD) and a C-terminalcatalytic fragment. To further assess the role of PKCd in H₂O₂-inducedcytochrome c release and apoptosis, we studied MCF-7 cells that stablyexpress the empty neo vector (MCF-7/neo) or the 35 kDa RD (MCF-7/PKCdRD)(see description of constructs in Example 2). In contrast to MCF-7/neocells, translocation of PKCd to mitochondria was attenuated inH₂O₂-treated MCF-7 cells stably expressing PKCdRD. The release ofcytochrome c in response to H₂O₂ was also attenuated in MCF-7/PKCdRD, ascompared to MCF-7/neo, cells. In concert with these results, H₂O₂treatment of MCF-7/neo cells was associated with the induction ofapoptosis and this response was attenuated in the MCF-7/PKCdRD cells.These findings demonstrate that targeting of PKCd to mitochondriacontributes to H₂O₂-induced cytochrome c release and apoptosis.

ROS have been implicated in the regulation of both cell growth andapoptosis. Although the signals activated by ROS are for the most partunclear, previous work has demonstrated that PKCd is phosphorylated ontyrosine in the cellular response to H₂O₂ treatment (see, e.g., Konishiet al. (1997) Proc. Natl. Acad. Sci. USA. 94, 11223-11237). Otherstudies have shown that c-Abl interacts with PKCd and is in partresponsible for tyrosine phosphorylation of PKCd in the response to H₂O₂(Sun et al. (2000) J. Biol. Chem. 275, 7470-7473). The availablefindings indicate that PKCd is activated by ROS and that PKCdphosphorylates and activates c-Abl. In a potential auto-catalytic loop,c-Abl phosphorylates and further activates PKCd. The present studiesdemonstrate that ROS induce targeting of PKCd to mitochondria and thatthis response is dependent on activation of the PKCd kinase function.The results also demonstrate that ROS-induced targeting of PKCd tomitochondria occurs in c-Abl^(−/−) cells. These findings indicate that,while c-Abl activation is dependent on PKCd, activation andtranslocation of PKCd to mitochondria in the response to H₂O₂ isindependent of the c-Abl kinase.

The results of the present studies show that ROS-induced cytochrome crelease is regulated by activation and translocation of PKCd tomitochondria. Taken together with the demonstration that c-Abl functionsin the apoptotic response to oxidative stress, these findings indicatethat signaling by both PKCd and c-Abl is needed for ROS-induced releaseof cytochrome c. Indeed, while treatment of c-Abl^(−/−) cells with H₂O₂is associated with PKCd activation and localization to mitochondria,these cells failed to respond to oxidative stress with release ofcytochrome c and induction of apoptosis. Finally, PKCd is also activatedby PDK1-mediated phosphorylation in the cellular response to serumstimulation (Le Good et al. (1998) Science 281, 2042-2045). Thus, PKCdappears to be functional in both pro and anti-apoptotic pathways andtherefore could represent a switch that determines cell fate.

Example 4 Targeting of the c-Abl Tyrosine Kinase to Mitochondria in theNecrotic Cell Death Response to Oxidative Stress

Cell culture: Human U-937 myeloid leukemia cells (ATCC, Manassas, Va.)were grown in RPMI 1640 medium supplemented with 10% heat-inactivatedfetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/mlstreptomycin and 2 mM L-glutamine. Wild-type, c-Abl^(−/−) and c-Abl+MEFs, MCF-7, MCF-7/c-Abl(K—R), SH-SY5Y (neuroblastoma) and 293T cellswere maintained in Dulbecco's modified Eagle's medium containing 10% FBSand antibiotics. Cells were treated with 1 mM H₂O₂ (Sigma) and 30 mMN-acetyl-L-cysteine (NAC; Sigma). Transient transfections were performedin the presence of calcium phosphate.

Immunofluorescence microscopy: Cells were plated onto poly D-lysinecoated glass coverslips 1 day prior to H₂O₂ treatment (1 hour) and thenfixed with 3.7% formaldehyde/PBS (pH 7.4) for 10 minutes. Cells werewashed with PBS, permeabilized with 0.2% Triton X-100 for 10 minutes,washed again and incubated for 30 minutes in complete medium. Thecoverslips were then incubated with 5 mg/ml of anti-c-Abl (K-12) for 1hour followed by Texas Red-goat anti-rabbit Ig (H+L) conjugate(Molecular Probes, Eugene, Oreg.). Mitochondria were stained with 100 nMMitotracker Green FM (Molecular Probes, OR). Nuclei were stained with4,6-diamino-2-phenylindole (DAPI; 1 mg/ml in PBS). Coverslips weremounted onto slides with 0.1 M Tris (pH 7.0) in 50% glycerol. Cells werevisualized by digital confocal immunofluorescence and images werecaptured with a cooled CCD camera mounted on a Zeiss Axioplan 2microscope. Images were deconvolved using Slidebook software(Intelligent Imaging Innovations, Inc., Denver, Colo.).

Isolation of mitochondria: Cells (3×10⁶) were washed twice with PBS,homogenized in buffer A (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mMHEPES, pH 7.4) and 110 mg/ml digitonin in a glass homogenizer (Pyrex no.7727-07) and centrifuged at 5000×g for 20 minutes. Pellets wereresuspended in buffer A, homogenized in a small glass homogenizer (Pyrexno. 7726) and centrifuged at 2000×g for 5 minutes. The supernatant wascollected and centrifuged at 11,000×g for 10 minutes. Mitochondrialpellets were disrupted in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mMNaCl, 1% NP-40, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mMsodium fluoride, 10 mg/ml leupeptin and aprotinin) at 4° C. and thencentrifuged at 15,000×g for 15 minutes. Protein concentration wasdetermined by the Bio-Rad protein estimation kit.

Preparation of cell lysates: Whole cell lysates (WCL) were prepared asdescribed (Kharbanda et al. (1995) Nature 376, 785-788) and analyzed forprotein concentration.

Immunoprecipitation and immunoblot analysis. Soluble proteins (100 mg)were incubated with anti-c-Abl (K-12; Santa Cruz Biotechnology) for 1hour and precipitated with protein A-sepharose for 30 minutes.Immunoprecipitates and lysates (5 mg) were resolved by SDS-PAGE andanalyzed by immunoblotting with anti-c-Abl (24-11; Santa Cruz),anti-HSP60 (Stressgen, Victoria, British Columbia), anti-b-actin (Sigma)anti-PCNA (Calbiochem), anti-PDGF receptor (Oncogene) and anti-PKCd(Santa Cruz).

Analysis of mitochondrial membrane potential: Cells were incubated with50 ng/ml Rhodamine 123 (Molecular Probes) for 15 minutes at 37° C. Afterwashing with PBS, samples were analyzed by flow cytometry using 488 nmexcitation and the measurement of emission through a 575/26 (ethidium)bandpass filter.

Quantitation of ATP: ATP levels were measured using an ATP DeterminationKit (Molecular Probes).

Assessment of apoptosis and necrosis by flow cytometry: Cells wereanalyzed by staining with annexin-V-fluorescein and propidium iodide(Annexin-V-FLOUS staining kit; Roche Diagnostics). Samples were analyzedby flow cytometry (Becton Dickinson) using 488 nm excitation and a 515nm bandpass filter for fluorescein detection and a >600 nm filter forpropidium iodide (PI) detection.

Results and Discussion:

To assess the effects of ROS on c-Abl, the subcellular localization ofc-Abl in response to H₂O₂ was investigates by measuring intracellularfluorescence with a high sensitivity CCD camera and image analyzer.Examination of the distribution of fluorescence markers in control MEFsshowed distinct patterns for anti-c-Abl (red signal) and amitochondrion-selective dye (Mitotracker; green signal). By contrast,exposure to H₂O₂ was associated with a marked change in fluorescencesignals (red and green→yellow/orange) supporting translocation of c-Ablto mitochondria. To confirm targeting of c-Abl to mitochondria in theresponse to ROS, mitochondria were isolated from MEFs treated with H₂O₂.Analysis of the mitochondrial fraction by immunoblotting with anti-c-Abldemonstrated an increase in c-Abl protein that was detectable at 30minutes and through 3 hours. Densitometric scanning of the signalsdemonstrated over a 5-fold increase in c-Abl protein at 0.5 to 1 hour ofH₂O₂ treatment. Immunoblotting for the mitochondrial HSP60 protein wasused to assess loading of the lanes. Moreover, purity of themitochondrial fraction was confirmed by reprobing the blots withantibodies against the cytoplasmic b-actin protein, the nuclear PCNAprotein and the cell membrane PDGF receptor. To estimate the amount ofc-Abl protein that localizes to mitochondria, whole cell andmitochondrial lysates, each prepared from 3×10⁶ cells, were subjected toimmunoblotting with anti-c-Abl. Densitometric scanning of the signalsand adjustment for lysate volume indicated that mitochondrial c-Abl isapproximately 4% of the total cellular c-Abl protein. Followingtreatment with H₂O₂, approximately 20% of total c-Abl localizes tomitochondria. As an additional control, mitochondrial lysates were firstsubjected to immunoprecipitation with anti-c-Abl. Immunoblot analysis ofthe immunoprecipitates with anti-c-Abl showed H₂O₂-induced increases inlevels of mitochondrial c-Abl protein. The demonstration that c-Abllevels are increased in the mitochondria of H₂O₂-treated human U-937leukemia cells and human neuroblastoma cells further indicated that thisresponse occurs in diverse cell types.

To confirm involvement of ROS in targeting of c-Abl to mitochondria,MEFs were treated with NAC, a scavenger of reactive oxygen intermediatesand precursor of glutathione. NAC treatment inhibited H₂O₂-inducedtranslocation of c-Abl to mitochondria. Also to determine whetherROS-induced activation of the c-Abl kinase function is necessary fortargeting of c-Abl to mitochondria, MCF-7 cells stably expressing akinase-inactive c-Abl(K—R) mutant at levels comparable that ofkinase-active c-Abl in MCF-7/neo cells were treated with H₂O₂, Thefinding that MCF-7/neo, but not MCF-7/c-Abl(K—R), cells respond to H₂O₂with translocation of c-Abl to mitochondria supported a requirement forthe c-Abl kinase function. These results indicate that ROS-induced c-Ablactivation is associated with the targeting of c-Abl to mitochondria.

As described in the examples above, ROS appears to activate c-Abl by amechanism dependent on the PKCd kinase. To determine whether PKCdcontributes to mitochondrial targeting of c-Abl, MEFs were treated withthe selective PKCd inhibitor, rottlerin. While rottlerin had no effecton constitutive levels of mitochondrial c-Abl, this agent inhibitedH₂O₂-induced c-Abl translocation. To extend the interaction of PKCd andc-Abl, 293 cells were cotransfected with HA-c-Abl and PKCd. Analysis ofthe mitochondrial fraction by immunoblotting with anti-HA demonstratedtargeting of HA-c-Abl to mitochondria is increased by H₂O₂ treatment. Bycontrast, cotransfection of HA-c-Abl and kinase-inactive PKCd wasassociated with less targeting of c-Abl to mitochondria and no apparenteffect of H₂O₂ treatment. These findings provide support for theinvolvement of ROS-induced activation of PKCd in mitochondrial targetingof c-Abl.

To determine whether c-Abl is necessary for ROS-induced loss ofmitochondrial transmembrane potential (Ψ), wild-type and c-Abl^(−/−)cells were treated with H₂O₂ and then stained with Rhodamine 123.Mitochondrial transmembrane potential was substantially diminished inH₂O₂-treated wild-type cells. By contrast, the Ψ was protected fromROS-induced loss in c-Abl^(−/−) cells, but not in c c-Abl^(−/−) cellstransfected to stably express c-Abl (c-Abl+). Cyclosporin A prevents thereduction in Ψ induced by various agents that open mitochondrialpermeability transition pores. In this context, pretreatment ofwild-type MEFs with cyclosporin A (100 mM for 1 hour) abrogated theH₂O₂-induced change in Ψ. Both apoptosis and necrosis are associatedwith decreases in Ψ, while necrosis is distinguished from apoptosis bydepletion of ATP and an early loss of plasma membrane integrity. Toassess the involvement of c-Abl in ROS-induced necrosis, wild-type,c-Abl^(−/−) and c-Abl+ cells were treated with H₂O₂ and assayed for ATPlevels. The results demonstrate that, while H₂O₂ treatment of wild-typeand c-Abl+ cells is associated with depletion of ATP, this response wasattenuated in c-Abl^(−/−) cells. As these findings support theinvolvement of c-Abl in a necrotic-like cell death, cells were stainedwith both annexin-V and PI to assess plasma membrane integrity. Theresults demonstrate that, compared to wild-type and c-Abl+ MEFs, loss ofplasma membrane integrity in response to H₂O₂ is attenuated inc-Abl^(−/−) cells. These findings demonstrate that ROS-induced targetingof c-Abl to mitochondria is associated with loss of mitochondrialmembrane potential, ATP depletion and necrotic cell death.

Activation of the c-Abl kinase in the cellular response to oxidativestress is dependent on PKCd and associated with release of mitochondrialcytochrome c. These findings provided support for involvement of c-Ablin the regulation of mitochondrial signaling. The present studiesdemonstrate that ROS target the c-Abl protein to mitochondria and thatthis response is dependent on the c-Abl kinase function. Moreover, inconcert with the demonstration that PKCd is required for ROS-inducedactivation of c-Abl, PKCd was shown to be necessary for targeting ofc-Abl to mitochondria. Importantly, localization of c-Abl tomitochondria is associated with loss of the mitochondrial transmembranepotential. Apoptosis and necrosis both involve loss of mitochondrialmembrane potential, while depletion of ATP is found selectively innecrosis. Thus, the demonstration that mitochondrial targeting of c-Ablis associated with depletion of ATP indicated that c-Abl is functionalin necrotic-like cell death. In this context, wild-type, but notc-Abl^(−/−), MEFs also responded to oxidative stress with dysfunction ofthe plasma membrane. These findings and the previous demonstration thatc-Abl is involved in ROS-induced cytochrome c release (Example 1)indicate that targeting of c-Abl to mitochondria confers bothpro-apoptotic and pro-necrotic cell death signals.

Example 5 Targeting of the c-Able Tyrosine Kinase to Mitochondria in ERStress-Induced Apoptosis

Cell culture: Rat1 cells and MEFs derived from wild-type and c-Abl^(−/−)mice were cultured in Dulbecco's modified Eagle's medium containing 10%heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/mlpenicillin and 100 mg/ml streptomycin. Cells were treated with A23187(Sigma) or Brefeldin A (Sigma).

Digital confocal immunofluorescence microscope: Cells grown onpoly-D-lysine coated glass coverslips were fixed (3.7% formaldehyde inPBS, pH 7.4; 10 minutes), permeabilized (0.2% Triton X-100; 10 min),blocked for 30 minutes in media containing serum. After rinsing withPBS, immunostaining was performed by incubating the cells with 50ng/slide of anti-c-Abl (K-12 rabbit polyclonal; Santa Cruz) andanti-grp-78 (C-20 goat polyclonal; Santa Cruz). For c-Abl andcalreticulin staining, cells were incubated first with anti-c-Abl alonefor 1 hour followed by blocking for 30 minutes in media containingserum. After washing with PBS, cells were then incubated withanti-calreticulin (rabbit polyclonal; StressGen). Finally, cells wereincubated with a 1:250 dilution of CY-3 or Fluorescein (FITC)-conjugatedanti-rabbit or anti-goat secondary antibodies (Jackson ImmunoResearch)for 1 hour. Mitochondria were stained with 0.006 ng/slide MitotrackerGreen FM (Molecular Probes). Nuclei were stained with4,6-diamino-2-phenylindole (DAPI; 1 mg/ml in PBS). Coverslips weremounted onto slides with 0.1 M Tris (pH 7.0) in 50% glycerol. Cells werevisualized by digital confocal immunofluorescence and images werecaptured with cooled CCD camera mounted on a Zeiss Axioplan 2microscope. Images were deconvolved using Slidebook software(Intelligent Imaging Innovations, Inc., Denver, Colo.).

Immnuo-electronemicroscopic analysis: Cells were fixed with 2%paraformaldehyde in 0.1 M sodium cacodylate buffer for 10 minutes,washed with three changes of cacodylate buffer, postfixed with 1% osmiumtetroxide for 5 minutes, dehydrated in graded ethanol, and infiltratedand polymerized with Poly/bed 812 overnight. Ultrathin sections were cutwith an ultramicrotome Nova (Leica). After etching with sodium periodatefor 10 minutes, the sections were rinsed with buffer and incubated withanti-c-Abl at a dilution of 1:10 overnight at 4° C. The sections wererinsed with buffer, incubated with protein A gold (15 nm) for 1 hour,rinsed again, and then fixed with 2% glutaradehyde in PBS for 2 minutes.After air drying, the sections were stained with 25 aqueous uranylacetate and with 0.5% lead citrate. The sections were examined andphotographed using a Hitachi H-600 electron microscope (Nessei Sagnyo)at 75 KV.

Isolation of the ER fraction: Cells were washed with PBS, lysed inhomogenization buffer (50 mM Tris-HCl, pH 8.0, 1 mM b-mercaptoethanol, 1mM EDTA, 0.32 M sucrose and 0.1 mM PMSF), and then centrifuged at5,000×g for 10 minutes. The supernatant was collected and centrifuged at105,000×g for 1 hour. The pellet was disrupted in lysis buffer (50 mMTris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM DTT, 1 mM sodiumorthovanadate, 1 mM PMSF, 10 mM sodium fluoride, 10 mg/ml leuptin andaprotinin) at 4° C. and then centrifuged at 15,000×g for 20 minutes. Theresulting supernatant was used as the ER fraction.

Isolation of cytoplasmic and nuclear fractions: The cytoplasmic andnuclear fractions were isolated as described in Example 1.

Isolation of mitochondria: Cells were washed twice with PBS, homogenizedin buffer A (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM HEPES, pH7.4) with 110 mg/ml digitonin in a glass homogenizer (Pyrex no. 7727-07)and then centrifuged at 5000×g for 5 minutes. Pellets were resuspendedin buffer A, homogenized in a glass homogenizer and centrifuged at1500×g for 5 minutes. The supernatant was collected and centrifuged at10,000×g for 10 minutes. Mitochondrial pellets were disrupted in lysisbuffer at 4° C. and then centrifuged at 15,000×g for 20 minutes. Proteinconcentration was determined by the Bio-Rad protein estimation kit.

Immunoblot analysis: Proteins were separated by SDS-PAGE, transferred tonitrocellulose and probed with anti-c-Abl (Calbiochem), anti-grp78(Santa Cruz), anti-calreticulin (StressGen), anti-HSP60 (StressGen),anti-b-actin (Sigma), anti-PCNA (Calbiochem) or anti-cytochrome c.Antigen-antibody complexes were visualized by enhanced chemiluminescence(ECL; Amersham Pharmacia Biotech).

Analysis of c-Abl activity: Cell lysates were prepared as described inExample 1 and subjected to immunoprecipitation with anti-c-Abl (K-12;Santa Cruz). The immunoprecipitates were resuspended in kinase buffer(see Example 1) containing 2.5 mCi of [g-32P]ATP and GST-Crk(120-225) orGST-Crk(120-212) for 15 minutes at 30° C. The reaction products wereanalyzed by SDS-PAGE and autoradiography.

Apoptosis assays: DNA content was assessed by staining ethanol-fixedcells with propidium iodide and monitoring by FACScan(Becton-Dickinson).

Localization of c-Abl to the ER: To assess the subcellular distributionof c-Abl, confocal microscopy was performed to detect colocalization ofc-Abl with proteins that are selectively expressed in differentorganelles. Using an antibody against the ER protein grp78, thedistribution of immunofluorescence was compared to that obtained withanti-c-Abl. Colocalization of grp78 (green) and c-Abl (red) wassupported by overlay of the signals (red+green→yellow/orange). Similarresults were obtained in colocalization studies of c-Abl and the ERprotein calreticulin. Immunogold labeling of cells with anti-c-Ablshowed expression of c-Abl in the cytoplasm, mitochondria and rough ER.These findings indicate that c-Abl localizes to the ER.

Subcellular fractionation studies were performed to define the fractionof c-Abl that associates with the ER. To assess intracellulardistribution of ER, cytosolic and mitochondrial fractions were subjectedto immunoblotting with anti-c-Abl. Analysis of equal amounts of proteinsfrom the fractions indicated that the concentration of c-Abl in the ERis higher than that found in the cytosol or mitochondria. The purity ofthe ER fraction was confirmed by immunoblotting with antibodies againstcalreticulin, b-actin and HSP60. Thus, the ER fraction includedcalreticulin, and little if any cytosolic b-actin or mitochondrialHSP60. Analysis of c-Abl protein in the different fractions, includingthe nucleus, indicated that c-Abl localized to the ER comprises about20% of c-Abl protein in the total cell lysate.

ER stress decreases ER-associated c-Abl: To assess whether ER stressaffects the subcellular localization of c-Abl, ER fractions wereisolated from cells treated with A23187. Immunoblot analysisdemonstrated that A23187 treatment is associated with a time-dependentdecrease in c-Abl levels. As shown previously, ER stress induced byA23187 was associated with increases in expression of grp78. Equalloading of the lanes was confirmed by immunoblotting withanti-calreticulin. ER fractions isolated from cells treated withBrefeldin A to inhibit transport of protein from the ER to Golgi werealso subjected to immunoblotting with anti-c-Abl. The resultsdemonstrate that Brefeldin A, like A23187, decreases c-Abl expression inthe ER. Brefeldin A treatment was also associated with increases ingrp78 and had little if any effect on levels of calreticulin. Thesefindings demonstrate that ER stress downregulates localization of c-Ablto the ER.

ER stress targets c-Abl to mitochondria: The subcellular relocalizationof c-Abl in response to ER stress was investigated by measuringintracellular fluorescence. Examination of the distribution offluorescence markers in control cells showed distinct patterns foranti-c-Abl (red signal) and a mitochondrion-selective dye (Mitotracker:green signal). By contrast, treatment with A23187 was associated with achange in fluorescence signals (red and green→yellow/orange) supportingtranslocation of c-Abl to mitochondria. Similar results were obtainedwith Brefeldin A-treated cells. By contrast, there was little if anychange in expression of c-Abl in the cytoplasm or nucleus. These resultsindicate that ER stress-induced downregulation of c-Abl in the ER isassociated with targeting of c-Abl to mitochondria.

ER stress activates the c-Abl kinase: To further define the distributionof c-Abl in response to ER stress, cytoplasmic and nuclear fractionsfrom A23187-treated cells were assessed by immunoblot analysis withanti-c-Abl. The results demonstrate that A23187 has little if any effecton c-Abl levels in the cytoplasm or nucleus. Purity of the fractions wasconfirmed by immunoblotting with anti-b-actin, anti-PCNA andanti-calreticulin. In contrast to the cytoplasm and nucleus, immunoblotanalysis of the mitochondrial fraction from A23187-treated cellsdemonstrated a time-dependent increase in c-Abl protein.

The mitochondrial fraction was also subjected to immunoprecipitationwith anti-c-Abl. Analysis of the immunoprecipitates for phosphorylationof GST-Crk(120-225) demonstrated that A23187 treatment is associatedwith increases in mitochondrial c-Abl activity. As a control, there wasno detectable phosphorylation of GST-Crk(120-212) that lacks the c-AblY-221 phosphorylation site. Densitometric scanning of the signalsobtained for phosphorylation of GST-Crk(120-225) as compared to thoseobtained for immunoprecipitated c-Abl protein indicated that A23187induces c-Abl activity.

Targeting of c-Abl to mitochondria was similarly assessed in cellstreated with Brefeldin A. Immunoblot analysis of the cytoplasmic andnuclear fractions showed no detectable effect of Brefeldin A on c-Abllevels. As found with A23187, analysis of the mitochondrial fractiondemonstrated Brefeldin A-induced increases in c-Abl protein. Inaddition, Brefeldin A treatment was associated with increases inmitochondrial c-Abl activity. Comparison of the signals found forGST-Crk(120-212) phosphorylation and c-Abl protein indicated thatBrefeldin A induces activation of the c-Abl kinase. These findings andthose obtained with A23187 demonstrate that ER stress is associated withtargeting of c-Abl to mitochondria and stimulation of c-Abl activity.

ER stress induces cytochrome c release and apoptosis by ac-Abl-dependent mechanism: To assess the functional significance of ERstress-induced targeting of c-Abl to mitochondria, wild-type andc-Abl^(−/−) MEFs were treated with A23187. Immunoblot analysis of themitochondrial fraction demonstrated A23187-induced increases inmitochondrial c-Abl levels in wild-type, but not c-Abl^(−/−), cells.Cytoplasmic fractions were also subjected to immunoblot analysis toassess release of mitochondrial cytochrome c. The results demonstratethat A23187 induces the release of cytochrome c in wild-type, but notc-Abl^(−/−), MEFs. Similar results were obtained in wild-type andc-Abl^(−/−) cells treated with Brefeldin A. In concert with thesefindings, A23187 treatment was associated with the induction of sub-G1DNA in wild-type cells, but had little effect on the induction ofapoptosis in c-Abl^(−/−) cells. The finding that ER stress-inducedapoptosis is also abrogated in c-Abl^(−/−) MEFs treated with Brefeldin Aprovided further support for involvement of c-Abl in this response.These results demonstrate that ER stress induces cytochrome c releaseand apoptosis by a c-Abl-dependent mechanism.

Stress signaling from the ER to mitochondria: The ER responds toalterations in homeostasis with the transduction of signals to thenucleus and cytoplasm. In this context, eukaryotic cells respond to theaccumulation of unfolded or excess proteins in the ER with i)transcriptional activation of genes encoding ER-resident proteins, andii) repression of protein synthesis. The ER-resident transmembranekinases, IRE1α/IRE1β, are activated by the presence of incorrectlyfolded proteins within the ER lumen and transduce signals that induceJNK/SAPK activity and gene transcription (see, e.g., Shamu et al. (1996)EMBO J. 15, 3028-3039). Inhibition of protein synthesis in the responseto unfolded proteins is signaled by the PERK transmembrane ER-residentkinase (Harding et al. (1999) Nature 397, 271-274). PERK has a lumenaldomain similar to that of IRE1 and a cytoplasmic kinase domain thatphosphorylates eIF2α. ER stress responses are also activated bydisruption of ER calcium homeostasis. The calcium ionophore A23187induces ER stress by increasing intracellular calcium pools. BrefeldinA, by contrast, induces ER stress by blocking transport of proteins fromthe ER to Golgi. Under conditions of excessive ER stress, cells activatesignaling pathways that induce apoptosis. However, the mechanismsresponsible for ER stress-induced apoptosis have been largely unknown.The results of the present studies demonstrate that the ER responds todiverse types of stress with the transduction of signals to mitochondriaand thereby the induction of apoptosis.

c-Abl confers ER stress signals to mitochondria: The available evidencehas shown that c-Abl is expressed in the nucleus and cytoplasm. Thepresent results demonstrate that c-Abl also localizes to the ER.Confocal microscopy studies demonstrate that c-Abl colocalizes with theER-associated grp78 and calreticulin proteins. Localization of c-Abl tothe ER was confirmed by immuno-electron microscopy and subcellularfractionation studies. Nuclear c-Abl is activated in the cellularresponse to genotoxic stress by mechanisms dependent on DNA-PK and ATM.Cytoplasmic c-Abl is activated in the response to oxidative stress by aPKCd-dependent mechanism (see, e.g., Example 1). The finding, asdescribed in Example 1, that c-Abl is required for the release ofcytochrome c in the oxidative stress response has further supported arole for c-Abl in targeting pro-apoptotic signals to mitochondria. Thepresent studies extend the link between c-Abl and cellular stress bydemonstrating that ER stress is associated with mitochondrial targetingof c-Abl. The results support a model in which ER stress inducestranslocation of the ER-associated c-Abl to mitochondria. The resultsalso support a functional role for c-Abl in transducing pro-apoptoticsignals that are activated by ER stress.

ER stress induces cytochrome c release and apoptosis by targeting c-Ablto mitochondria: In the cytosol, cytochrome c associates with a complexof Apaf-1 and caspase-9, and thereby induces the activation of caspase-3(see, e.g., Li et al. (1997) Cell 91, 479-489). The induction ofapoptosis is associated with caspase-3-mediated cleavage ofpoly(ADP-ribose) polymerase (PARP), PKCd and other proteins. While ERstress can induce apoptosis, the involvement of cytochrome c release inthis response has been unknown. In the present studies, the finding thatER stress induces the release of mitochondrial cytochrome c providedfurther support for signaling from the ER to mitochondria. Importantly,the induction of cytochrome c release by ER stress was attenuated inc-Abl^(−/−) cells. Moreover, c-Abl^(−/−) cells were defective in theapoptotic response to ER stress. These findings indicate that ERstress-induced cytochrome c release and apoptosis are mediated bytargeting c-Abl from the ER to mitochondria.

Example 6 Proteosome Degradation of Catalase is Regulated by the c-Abland Arg Tyrosine Kinases

c-Abl and Arg regulate catalase stability: To determine whether c-Abland/or Arg regulates catalase expression, lysates from wild-type,c-abl−/− and arg−/− MEFs were analyzed by immunoblotting with ananti-catalase antibody. The results demonstrated little if any effect ofc-Abl or Arg deficiency on catalase levels. By contrast, catalaseexpression was increased 2-3-fold in c-abl−/−arg−/− cells. The findingthat similar results were obtained in separate clones of c-abl−/−arg−/−cells indicated that the increase in catalase expression was not due toclonal variation.

To confirm that the absence of Arg is associated with increases incatalase, the c-abl−/−arg−/− cells were transduced with a retrovirusexpressing Arg. Compared to c-abl−/−arg−/− cells expressing the emptyvector, Arg expression was associated with decreased levels of catalase.

Catalase mRNA levels were found to be similar in wild-type MEFs andc-abl−/−arg−/− cells, indicating that catalase is regulated by stabilityof the protein. To assess the half-life of catalase, MEFs andc-abl−/−arg−/− cells were treated with CHX and then monitored forcatalase levels. The results demonstrated a catalase half-life ofapproximately 10.5 hours in MEFs and greater than 24 hours inc-abl−/−arg−/− cells. To confirm the effects of c-Abl and Arg oncatalase stability, 293 cells were transfected to express Flag-catalaseand c-Abl or Arg. Cotransfection of Flag-catalase and the empty vectorresulted in a half-life of greater than 9 hours. By contrast, thehalf-life of catalase was less than 3 hours when co-expressed with c-Ablor Arg. These findings demonstrate that the stability of catalase isregulated by c-Abl and Arg.

Ubiquitination of catalase by a c-Abl/Arg-mediated mechanism: Todetermine if catalase is subject to ubiquitination, catalase wasincubated in an in vitro ubiquitination system. Analysis of reactionproducts by immunoblotting with an anti-catalase antibody demonstratedreactivity over a range of electrophoretic mobilities. In concert withthese findings, immunoblotting with an anti-ubiquitin (Ub) antibodydemonstrated ubiquitination of catalase.

To determine if catalase is regulated by the Ub-proteosome pathway,anti-catalase antibody immunoprecipitates from wild-type MEFs wereanalyzed by immunoblotting with an anti-Ub antibody. The results showedanti-Ub reactivity over a range of electrophoretic mobilities. Bycontrast, similar studies in c-abl−/−arg−/− cells demonstrated asubstantial decrease in anti-Ub reactivity. As a control,immunoprecipitation of catalase was found to be comparable from thewild-type MEFs and c-abl−/−arg−/− cells. In the reciprocal experiment,anti-Ub immunoprecipitates analyzed by immunoblotting with ananti-catalase antibody demonstrated a higher level of ubiquitinatedcatalase in MEFs as compared to c-abl−/−arg−/− cells. Studies ofc-abl−/− and arg−/− cells also demonstrated higher levels ofubiquitinated catalase. These results demonstrate that catalase isubiquitinated by a c-Abl/Arg-dependent mechanism.

Degradation of catalase by the 26S proteosome: In studies using an invitro Ub-proteosome system, catalase was found to be subject toproteosomal degradation. As a control, degradation was inhibited when[g-S]ATP was substituted for ATP to prevent ubiquitination. Degradationof catalase was also blocked when the proteosome inhibitor, MG132, wasadded to the reaction. Lactacystin, another proteosome inhibitor, wasused to assess degradation of catalase in cells. Treatment of wild-typeMEFs with lactacystin was associated with an increase in catalaselevels. Lactacystin treatment of c-abl−/−arg−/− cells, however, hadlittle if any effect.

In 293 cells expressing Flag-catalase and c-Abl, lactacystin increasedthe half-life of catalase. Similar results were obtained withlactacystin when Flag-catalase was coexpressed with Arg. MG132 alsoincreased the half-life of catalase in cells. These findings indicatethat c-Abl and Arg target catalase for ubiquitination and proteosomaldegradation.

c-Abl and Arg phosphorylate catalase: To assess the potential role ofc-Abl and Arg in regulating catalase, studies were performed todetermine whether catalase is subject to tyrosine phosphorylation.Analysis of anti-Flag immunoprecipitates with anti-P-Tyr demonstratedtyrosine phosphorylation of catalase. Moreover, a similar analysis inthe presence of MG132 showed that the ubiquitinated forms of catalaseare phosphorylated on tyrosine.

To define potential phosphorylation sites, catalase was incubated withc-Abl and then subjected to tryptic digestion. Analysis of the fragmentsby HPLC separation and Edmon sequencing demonstrated phosphorylation ofY231 and Y386. Compared to wild-type catalase, mutation of Y231 to Fresulted in a decrease in c-Abl-mediated tyrosine phosphorylation. Asimilar decrease in tyrosine phosphorylation was observed with thecatalase(Y386F) mutant. The results also show that, in the presence ofMG132, Arg-mediated tyrosine phosphorylation of ubiquitinated catalaseis decreased for the Y231F and Y386F mutants. These findings indicatethat catalase is phosphorylated on Y231 and Y386 by c-Abl and Arg andthat these modifications are necessary for ubiquitination of catalase.

Ubiquitination and stability of catalase are regulated by c-Abl/Argphosphorylation: To determine whether catalase stability is directlyregulated by c-Abl and Arg, expression of Flag-tagged catalase wascompared to that of Flag-catalase(Y231F) and Flag-catalase(Y386F).Levels of the two Y→F mutants were higher than that found with wild-typecatalase. Moreover, mutation of the PFNP motif to abrogate c-Abl and Argbinding resulted in increased catalase expression. In concert with theseresults, stability of catalase was increased by mutation of the Y231,Y386 or P293 sites. Ubiquitination of catalase(Y231F) andcatalase(Y386F) was also substantially decreased compared to that ofwild-type catalase. Similar results were obtained with thecatalase(P293A) mutant.

To further define the effects of c-Abl, anti-Flag immunoprecipitateswere analyzed from cells expressing Flag-catalase and Myc-tagged c-Abl.Ubiquitination of wild-type Flag-catalase was increased by c-Abl. Bycontrast, c-Abl had little effect on ubiquitination of the catalasemutants. Arg also increased ubiquitination of wild-type catalase, butnot the Y→F or P→A mutants. These results demonstrate that tyrosinephosphorylation of catalase by c-Abl and Arg regulates catalaseubiquitination and stability.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1-32. (canceled)
 33. A method for identifying a modulator of catalaseubiquitination, the method comprising: a) contacting a polypeptidecomprising catalase to ubiquitin in the presence of a test compound; b)measuring the ubiquitination of the polypeptide in the presence of thetest compound, wherein altered ubiquitination of the polypeptide in thepresence of the test compound compared to the absence of the testcompound indicates that the compound is a modulator of catalaseubiquitination.
 34. A method for identifying a modulator of catalasephosphorylation, the method comprising: a) contacting a polypeptidecomprising catalase to a test compound; b) measuring phosphorylation ofthe polypeptide in the presence of the test compound, wherein alteredphosphorylation of the polypeptide in the presence of the test compoundcompared to the absence of the test compound indicates that the compoundis a modulator of catalase phosphorylation. 35-38. (canceled)